Techniques which we have recently developed for the construction of viable nuclear-cytoplasmic hybrid cells by nuclear transplantation will be used to investigate the role of the cytoplasm in cell differentiation and transformation. Cytoplasts (that is, enucleated cells) and karyoplasts (that is, nuclei surrounded by a thin shell of cytoplasm and an outer cell membrane) prepared from various differentiated and transformed cell lines which exhibit distinct morphological and/or biochemical traits will be fused to form true cytoplasmic-nuclear hybrid cells. The hybrids and their progeny will then be analyzed for the maintenance, loss, or acquisition of the parent cell traits. Construction of the appropriate hybrid lines will permit direct investigation of such areas as cytoplasmic control of neuronal differentiation, immunoglobulin synthesis and hormone dependent growth; the requirement of an integrated viral genome for maintenance of expression of various traits of transformed cells; and the role of the cytoplasm in carcinogen-induced malignancy. In effect, then, the major goal of the proposed work is to apply newly developed techniques to the creation of novel systems for investigating gene expression and its control in mammalian cells. We have also found that a significant portion of karyoplasts can regenerate cytoplasm to reform whole viable cells. Continuation of this work should lead to fruitful investigations of the biogenesis and functions of various cellular organelles and of the regulatory mechanisms which govern generation of cytoplasmic mass and coordinate nuclear events with cytoplasmic growth. Moreover, it appears that some cell lines derived from regenerated karyoplasts are markedly altered in certain aspects of growth control. Analysis of such lines may permit new insight into the changed growth pattern which accompany transformation.